interest.Data and materials availability:The cryo-EM maps
and coordinates have been deposited to the Electron
Microscopy Data Bank and Protein Data Bank under the
accession numbers listed in table S3. Materials generated in
this study will be made available on request after signing a
materials transfer agreement with Vir Biotechnology or the
University of Washington. This work is licensed under a Creative
Commons Attribution 4.0 International (CC BY 4.0) license,
which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited. To
view a copy of this license, visit https://creativecommons.org/
licenses/by/4.0/. This license does not apply to figures/
photos/artwork or other content included in the article that is
credited to a third party; obtain authorization from the rights
holder before using such material.SUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.abl8506
Materials and MethodsFigs. S1 to S7
Tables S1 to S4
References ( 84 – 103 )
MDAR Reproducibility Checklist11 August 2021; accepted 2 November 2021
Published online 9 November 2021
10.1126/science.abl8506CORONAVIRUS
Rapid assessment of SARS-CoV-2Ðevolved variants
using virus-like particles
Abdullah M. Syed1,2†, Taha Y. Taha^3 †, Takako Tabata^3 †, Irene P. Chen3,4, Alison Ciling^2 , Mir M. Khalid^3 ,
Bharath Sreekumar^3 , Pei-Yi Chen^3 , Jennifer M. Hayashi^3 , Katarzyna M. Soczek2,5,
Melanie Ott2,3,6, Jennifer A. Doudna1,2,5,7,8,9,10,11
Efforts to determine why new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants
demonstrate improved fitness have been limited to analyzing mutations in the spike (S) protein with
the use of S-pseudotyped particles. In this study, we show that SARS-CoV-2 virus-like particles
(SC2-VLPs) can package and deliver exogenous transcripts, enabling analysis of mutations within all
structural proteins and at multiple steps in the viral life cycle. In SC2-VLPs, four nucleocapsid (N)
mutations found universally in more-transmissible variants independently increased messenger RNA
delivery and expression ~10-fold, and in a reverse genetics model, the serine-202→arginine (S202R) and
arginine-203→methionine (R203M) mutations each produced >50 times as much virus. SC2-VLPs provide
a platform for rapid testing of viral variants outside of a biosafety level 3 setting and demonstrate
N mutations and particle assembly to be mechanisms that could explain the increased spread of variants,
including B.1.617.2 (Delta, which contains the R203M mutation).
T
he COVID-19 pandemic is a leading cause
of death globally, owing to the ongoing
emergence of severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2)
variants with increased transmissibility.
Understanding the molecular determinants of
enhanced infectivity is central to vaccine and
therapeutic development, but research is hin-
dered because SARS-CoV-2 can be studied
only in a biosafety level 3 (BSL-3) laboratory.
Furthermore, technical challenges impede ef-
forts to generate mutant infectious clones of
SARS-CoV-2 ( 1 – 5 ). Current studies employ
spike (S) protein–pseudotyped lentivirus sys-
tems for evaluation of S-mediated ACE2 re-
ceptor binding and cell entry ( 6 , 7 ). However,
most mutations in circulating variants occur
outsideoftheSgeneandarethusinaccessible
by this approach ( 8 ).
All SARS-CoV-2 variants of interest or con-
cern defined by the World Health Organiza-
tion contain at least one mutation with >50%
penetrance within a seven–amino acid span
(199 to 205) in the nucleocapsid (N) protein,
which is required for replication, RNA bind-
ing, packaging, stabilization, and release ( 8 ).
Despite its functional importance and em-
ergence as a mutational hotspot, the N protein
has not been widely studied because of the
absence of simple and safe cell-based assays.
Biochemical analysis of N has also proven dif-
ficult because of its instability and propensity
to assemble or phase-separate and to bind RNA
nonspecifically ( 9 – 11 ). To investigate N func-
tion,effectsofmutations,andotheraspectsof
SARS-CoV-2 biology, we set out to develop a
system to package and deliver exogenous RNA
transcripts into cells by means of virus-like
particles (VLPs).
We reasoned that a process that mimics
viral assembly to package and deliver reporter
transcripts would simplify the analysis of suc-
cessful virus production, budding, and entry.
Previous studies have shown that coexpres-sion of only the structural proteins of corona-
viruses generates VLPs that contain all four
structural proteins ( 12 – 17 ). These VLPs appear
to have similar morphology to infectious viruses
and have been proposed as vaccine candidates
( 18 ). A key requirement for such VLPs to deliver
reporter transcripts into cells is the recogni-
tion of a cis-acting RNA sequence that triggers
packaging. During viral assembly, the N pro-
tein is thought to recognize one or more RNA
structures within open reading frames 1a
and 1b (ORF1ab), thus enabling the full viral
genome that contains this sequence to be
packaged to the exclusion of viral subgenomic
and host transcripts ( 19 ). The identification of
such a SARS-CoV-2 cis-acting RNA element is
required to create SARS-CoV-2 VLPs (SC2-VLPs)
that incorporate and deliver engineered tran-
scripts by this mechanism.
On the basis of the reported packaging se-
quences for related viruses, including murine
hepatitis virus and severe acute respiratory
syndrome coronavirus (SARS-CoV), we hypo-
thesized that the SARS-CoV-2 packaging sig-
nal might reside within a region termed“T20”
(nucleotides 20080 to 22222) that encodes
nonstructural proteins 15 and 16 (nsp15 and
nsp16) (Fig. 1A) ( 16 , 19 – 21 ). We designed a
transfer plasmid that encodes a luciferase
transcript containing T20 within its 3′un-
translated region (UTR). We tested for SC2-
VLP production by cotransfecting the transfer
plasmid into packaging cells (293T cells) along
with plasmids that encode the viral structural
proteins (Fig. 1B). Supernatant collected from
these cells was filtered and incubated with
receiver 293T cells that coexpress SARS-CoV-2
entry factors ACE2 and TMPRSS2 (Fig. 1B).
We observed luciferase expression in receiver
cells only in the presence of all structural
proteins (S, M, N, and E) as well as the T20-
containing reporter transcript (Fig. 1C). Sub-
stituting any one of the structural proteins or
the luciferase-T20 transcript with a luciferase-
only transcript decreased luminescence in
receiver cells >200- and 63-fold, respectively
(Fig. 1C). We also conducted this experiment
using Vero E6 cells that endogenously express
ACE2 and once again observed robust lucifer-
aseexpressiononlyinthepresenceofallfive
components (fig. S1A).
VLP-mediated transcript delivery required
untagged native M protein and a low ratio1626 24 DECEMBER 2021•VOL 374 ISSUE 6575 science.orgSCIENCE
(^1) Gladstone Institute of Data Science and Biotechnology, San
Francisco, CA, USA.^2 Innovative Genomics Institute, University
of California, Berkeley, Berkeley, CA, USA.^3 Gladstone Institute
of Virology, San Francisco, CA, USA.^4 Biomedical Sciences
Graduate Program, University of California, San Francisco, CA,
USA.^5 Department of Molecular and Cell Biology, University of
California, Berkeley, CA, USA.^6 Department of Medicine,
University of California San Francisco, CA, USA.^7 Molecular
Biophysics and Integrated Bioimaging Division, Lawrence
Berkeley National Laboratory, Berkeley, CA, USA.^8 Howard
Hughes Medical Institute, University of California, Berkeley,
Berkeley, CA, USA.^9 Department of Chemistry, University of
California, Berkeley, Berkeley, CA, USA.^10 California Institute for
Quantitative Biosciences (QB3), University of California,
Berkeley, Berkeley, CA, USA.^11 Gladstone-UCSF Institute of
Genomic Immunology, San Francisco, CA, USA.
*Corresponding author. Email: [email protected] (J.A.D.);
[email protected] (M.O.)
†These authors contributed equally to this work.
RESEARCH | REPORTS